CN105085672B

CN105085672B - 3D protein specific monoclonal immunoglobulin A antibodies and compositions thereof

Info

Publication number: CN105085672B
Application number: CN201510026683.2A
Authority: CN
Inventors: 鄢慧民; 李么明; 周谛晗; 赵巴利
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-01-19
Filing date: 2015-01-19
Publication date: 2020-03-13
Anticipated expiration: 2035-01-19
Also published as: CN105085672A

Abstract

The present invention provides 3D protein specific monoclonal immunoglobulin a antibodies and compositions thereof that specifically bind to a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and SEQ ID NO: one consensus sequence in group 2. The invention also provides a composition comprising at least one first 3D protein-specific monoclonal immunoglobulin a antibody that specifically binds to a protein encoded by a consensus sequence of SEQ ID NO: 1, at least one second 3D protein-specific monoclonal immunoglobulin a antibody, specifically binding to a monoclonal antibody represented by the consensus sequence SEQ ID NO: 2, and a pharmaceutically acceptable solution.

Description

3D protein specific monoclonal immunoglobulin A antibodies and compositions thereof

Technical Field

The present invention relates generally to prophylactic and therapeutic agents for enteroviruses, and more particularly to monoclonal immunoglobulin a (iga) antibodies specific for the 3D protein of enteroviruses, and further to prophylactic or therapeutic compositions comprising the 3D protein specific monoclonal immunoglobulin a antibodies.

Background

Enteroviruses are divided into four major subtypes, a, B, C and D, each of which contains many serotypes. Enteroviruses cause different diseases. For example, the major pathogens in hand, foot and mouth disease (HFMD) are enterovirus type 71 (EV 71) and Coxsackievirus (CV) belonging to the picornavirus family. The hand-foot-and-mouth disease threatens public health increasingly, especially for infants. EV71 and CV infections cause severe aseptic meningitis, encephalitis, myocarditis, acute paralysis, pulmonary edema, leading to high mortality.

As a member of the enterovirus genus within the picornavirus family, EV71 has a typical positive-sense single-stranded RNA genome, containing a single open reading frame, encoding 4 capsid proteins (VP 1-4) and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). 3D (also referred to as 3D)^pol) The protein, as a viral RNA-dependent RNA polymerase (RdRp), plays a major role in viral negative strand synthesis and uridylation (uridylation) of some proteins. From studies of the crystal structure of EV71, it was found that one EV71 virion comprises a nucleocapsid formed of 60 copies of the three viral structural proteins VP1-VP3, to the inner surface of which 60 copies of the small protein VP4 are attached.

3D has high sequence homology in all enteroviruses, but low homology to human proteins. The 3D of EV71 has structural/sequence similarity to homologous RdRps of polioviruses of the picornavirus family, coxsackieviruses, rhinoviruses and foot and mouth disease virus polymerases.

3D has an N-terminal active site. Kiener et al used recombinant 3CD protein from strain EV 71C 4 as an immunogen, isolated monoclonal antibody 4B12 (IgG 1), recognized the linear epitope DFEQALFS (corresponding to positions 53-60 of 3D and 1784-1791 of EV71 polyprotein), near the active site of 3D polymerase, 4B12 all enterovirus 71 subtypes detected by denaturation point hybridization (Kiener et al, Characterization of a monoclonal antibody biosynthesis the 3D polymerase of enterovirus 71 and its use for the detection of human enterovirus A infection.J Virol Methods.2012, 180(1-2): 75-83). 4B12 is capable of detecting a wide range of subtype strains, with the potential to suggest that 3D-specific monoclonal antibodies may be useful for diagnosing infection, but no mention or suggestion is made to use 4B12, let alone 3D-specific antibodies in general, prophylactically or therapeutically.

Since vaccines are the best strategy for controlling infectious diseases, different candidate vaccines for EV71 were investigated, including EV71 inactivated whole virus vaccines, live attenuated virus vaccines, recombinant VP1 vaccines, VP 1-based DNA vaccines, synthetic peptide vaccines and virus-like particle vaccines. Formalin inactivated EV71 vaccine in miceAnd rhesus monkeys elicited satisfactory immunoprotection and cross-reactive neutralizing antibodies, and in china, the EV71 inactivated vaccine had completed a three-phase clinical trial in 2013. Since the outer capsid contains the major antigenic site during infection, the synthetic peptide vaccine contains only nucleocapsid proteins, including VP1 and VP2 (Kung et al. update of the later of infectious agents 71 vaccines.Expert Opin Biol Ther.2014;3:1-10）。

Since EV71 is an RNA virus, replication of, for example, enterovirus type 71 nucleocapsid genes by error-prone RdRp may result in considerable genetic and antigenic diversity. Chen et al demonstrated that the genotype does not reflect its antigenicity by a panel of monoclonal antibodies against VP1, and that the EV71 virus can be divided into different antigenic groups (Chen et al, antigenic catalysis of divergent genes man Enterovirus 71 viruses by a panel of healthy pathogenic antigens: current genetic typing of EV71 does notrefect.Vaccine.2013, 31 (425-30) and (2). All this suggests that it is a challenge to select an ideal strain to develop a vaccine with broad effectiveness.

Because of the current absence of vaccines and specific antiviral drugs, intravenous immunoglobulin (IVIG) injection in humans has been used clinically to treat severe EV71 infection. However, the finding of antibody-dependent potentiation (ADE) in EV71 infection shows a combined effect of the antibody in controlling EV71 infection. ADE is a phenomenon in which pre-existing sub-levels of neutralizing antibodies enhance viral entry and replication. Sub-levels of neutralizing antibodies showed that EV71 was boosted to infect Fc receptor-containing human monocytes and EV 71-infected mice were exacerbated. Furthermore, the extensive cross-reactivity that exists between enterovirus antibodies may also be a potential risk for ADE in EV71 infection.

Han et al demonstrated that low concentrations (50. mu.g/ml) of neutralizing antibodies (immunoglobulins) against EV71 enhanced enterovirus type 71 infected monocyte cell lines (Han et al, Antibody dependent infection of infectious disease 71in vitroandin vivo.Virol J.2011; 8: 106). Cao et al further investigated the role of each IgG subclass in neutralizing and enhancing EV71 infectionAnd found that the neutralizing activity of Human immunoglobulin is mainly mediated by IgG1 subclass, the IgG2 subclass is low, the IgG3 part has no neutralizing activity, but the infection of enterovirus 71 Type in vitro is improved (Cao et al, Human IgG sublases against infectious disease Type 71: neutrallized adjuvant treatment of infection.PLoS One.2013, 8 (e 64024) in the formula (5). Thus, the design of a novel vaccine should induce the production of antibodies with strong neutralization but weak ADE activity.

Two general IgG monoclonal antibodies, one anti-VP 1 (Lim et al, chromatography of an isolated-dependent monoclonal antibody against obtained in line with a random mutagenesis approach for the expression of infectious 71 infection) have been known so far.PLoS One.2012, (7) (1) e 29751), another anti-VP 3 (Kiener et al. A novel unsaturated monoclonal antibody against antigen expressed in virus 71 which targets the high level conserved "knob" region of VP3 protein. PLoS Negl Trop Dis. 2014, 8(5) e 2895. However, the ADE capacity of these universal antibodies was not investigated.

The studies by Jia et al raised a serious concern for the safety of inactivated EV71 virus vaccines (Jia et al. the cross-reactivity of the enterovirus 71 to human vaccine tissue and reactivity of the cross-reactive fragments).Virol J.2010, 7: 47). Jea et al demonstrated the presence of specific IgG in the serum of patients with EV71 infection, with cross-reactivity towards human brain, followed by the preparation of polyclonal serum using 19 purified polypeptides, antisera to P230-323, P646-755, P857-1012 and P1329-1440 showed strong staining of neurons in adult brain and fetal medulla, antisera to P1-69, P324-443, P444-565, P566-665, P746-876, P1441-1526, P1549-1668, P1732-1851 and P2072-2193 showed weak staining, and antisera to P70-159, P140-249, P1197-1338, P1649-1731 and P1843-1951 showed no staining.

Therefore, there is an urgent need to develop new means to deal with the hand-foot-and-mouth disease caused by enteroviruses such as EV71 and CV.

The invention screens rapidly to achieve the purposes of reducing analysis cost and improving inspection efficiency.

Disclosure of Invention

In one embodiment, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, or a consensus sequence.

In another embodiment of the 3D protein specific monoclonal immunoglobulin a antibody, the antibody of SEQ ID NO: the 3D protein specific monoclonal immunoglobulin A antibody combined with the polypeptide shown in 1 is a hybridoma cell strain 3D-2A10-IgA (CCTCC NO: C2014144) and has a sequence similar to that shown in SEQ ID NO: 2 is hybridoma cell strain 3D-3A12-IgA (CCTCC NO: C2014142).

In another embodiment of the 3D protein specific monoclonal immunoglobulin a antibody, the amino acid sequence of SEQ ID NO: 1 is a polypeptide selected from the group consisting of SEQ ID NOS: 3-8, SEQ ID NO: 2 is selected from the group consisting of SEQ ID NOS: 10-14.

The invention also provides the composition. In one embodiment, the composition comprises at least one first 3D protein-specific monoclonal immunoglobulin a antibody that specifically binds to a protein encoded by the consensus sequence SEQ ID NO: 1, at least one second 3D protein-specific monoclonal immunoglobulin a antibody, specifically binding to a monoclonal antibody represented by the consensus sequence SEQ ID NO: 2, and a pharmaceutically acceptable solution.

In another embodiment of the composition, the peptide has a sequence identical to SEQ ID NO: 1 is a hybridoma cell strain 3D-2A10-IgA (CCTCC NO: C2014144) which is similar to the monoclonal immunoglobulin A antibody of SEQ ID NO: 2 is hybridoma cell strain 3D-3A12-IgA (CCTCC NO: C2014142).

In another embodiment of the composition, the first polypeptide is a polypeptide selected from the group consisting of SEQ ID NOS: 3-8, and the second polypeptide is a polypeptide selected from the group consisting of SEQ ID NOS: 10-14.

The objects and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings.

Drawings

Preferred embodiments of the present invention will now be described with reference to the drawings, wherein like reference numerals represent like elements.

Figure 1, characterization of EV 713D specific monoclonal IgG antibodies. The panel (A) shows indirect immunofluorescent staining of EV71 infected cells with 3D-specific IgG monoclonal antibodies (3A 12, 2A10, 7A6G1 and 11F 1), flagellin-specific monoclonal antibody (5G 10) as negative control and 3D-immunized mouse serum as positive control. (B) immunoblotting of lysates of EV71 infected VERO-1008 cells with 3D-specific IgG monoclonal antibodies (3A 12, 2A10, 7A6G1 and 11F 1), flagellin-specific monoclonal antibodies (5G 10) as negative control and 3D-immunized mouse serum as positive control.

Figure 2, 3D specific IgG monoclonal antibodies inhibit EV71 replication in cells. (A) shows the viral titer in the presence of intracellular antibodies via transfection. (B) graphical representation of viral titers in the presence of different doses of 2A10 or EV-5. EV-5 is an EV71VP2 specific monoclonal antibody.

FIG. 3, 3D specific IgG monoclonal antibodies inhibit 3D polymerase activity in vitro. (A) schematically illustrates 3D (of RdRp) mediated RNA extension. The (B) panel shows the effect of different IgG monoclonal antibodies on 3D-mediated RNA elongation.

Figure 4, graphical representation of the antiviral function of EV 713D specific IgG monoclonal antibodies in a murine model.

FIG. 5, a graph showing antibody-dependent enhancement of EV71 replication in the presence of 2A10-IgG or EV-5-IgG monoclonal antibody.

Fig. 6, EV 713D specific epitopes of 3a12 and 2a10, steric states in the three-dimensional model of EV 713D (1 RA 6). EV 713D (1 RA 6) was used to indicate the position of the two recognition epitopes of 3a12 and 2a 10.

Figure 7, identification of EV 713D specific 3D protein specific monoclonal immunoglobulin a antibodies. The panel (A) shows indirect immunofluorescent staining of EV71 infected cells with 3D specific 3D protein specific monoclonal immunoglobulin A antibodies 3A12-IgA and 2A10-IgA, 16CF7IgA (anti-MeV) as negative control and sera from 3D immunized mice as positive control. (B) EV71 infected VERO-1008 cells (WB), 3D protein specific monoclonal immunoglobulin A antibodies used were 3A12-IgA and 2A10-IgA, 16CF7IgA as negative controls, and 3D protein immunized mice sera as positive controls.

Figure 8, 3D protein specific monoclonal immunoglobulin a antibody inhibits EV71 replication intracellularly. (A) The viral titers in EV 71-infected polarized VERO-pIgR cells in the presence of IgA antibodies at the base of the Transwell are shown. (B) The viral titers in EV71 infected polarized VERO-pIgR cells in the presence of different doses of 3A12-IgA or 16CF7IgA at the basal level are shown. (C) The viral titers in EV 71-infected polarized VERO-pIgR cells in the presence of different IgG antibodies at the base are shown. (D) The viral titers in EV71 infected polarized VERO-1008 cells in the presence of different IgA antibodies at the base are shown.

FIG. 9, EV 713D-IgA inhibited viral protein accumulation. (A) Immunoblotting (WB) detected the expression of 3D and VP2 in EV71 infected polarized VERO-pIgR cells, with the presence of IgA antibodies at the base during culture. (B) ELISA results are shown, and expression of 3D and VP2 in cells as described in (A) is examined.

FIG. 10, graphical representation of the effect of different IgA monoclonal antibodies on 3D mediated RNA extension.

Figure 11, graphical representation of the antiviral effect of EV 713D protein specific monoclonal immunoglobulin a antibodies in a mouse model.

FIG. 12, is a schematic representation of a 3D immunoblot expressed from a VTT-3D expression vector (VTT-3D, an attenuated vaccinia virus recombinantly expressing 3D).

Figure 13, illustrates immunization and challenge procedure.

Figure 14, graphical representation of 3D specific antibody responses after priming: (A) serum IgG, (B) serum IgA, (C) salivary IgA, and (D) vaginal IgA.

Figure 15, graphical representation of 3D specific antibody responses after the first boost: (A) serum IgG, (B) serum IgA, (C) salivary IgA, and (D) vaginal IgA.

Figure 16, graphical representation of 3D specific antibody responses after the second boost: (A) serum IgG, (B) serum IgA, (C) salivary IgA, and (D) vaginal IgA.

Figure 17, graphical representation of the 3D specific IgG antibody response in the intestine of neonatal mice born to immunized mothers.

Figure 18, is a graph showing the percent survival of neonatal mice challenged with mouse adapted strain EV 71.

FIG. 19, is a graph showing antibody responses after immunization of BALB/c mice with purified 3D protein.

Detailed Description

The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention.

Throughout this application, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Unless otherwise indicated, the practice of the present invention will employ, within the skill of the art, techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry and immunology.

These techniques have been fully explained in the literature, such as molecular cloning: laboratory instructions, third edition (Sambrook and Russel, 2001), animal cell culture (RI Freshmey eds., 1987), Current protocols in molecular biology (FMAusubel et al eds., 1987, including supplementary to 2001), Current protocols in immunology (JE Coligan et al eds., 1991), A Manual of immunoassay "(D.Wild eds., Stokes Press, New York, 1994), methods of immunological analysis (R.Masseyeff, WH Albert and NA Staines et al eds., Weinheim: VCH VERLAGS GESELLSCHAFT MBH, 1993).

The invention discovers that the 3D protein specific monoclonal immunoglobulin A antibody can effectively inhibit enterovirus 71 (EV 71) in a Transwell model, and more importantly, can protect a host from virus attack.

The 3D protein (SEQ ID NO: 15) of the EV71 BrCr strain was expressed and purified, and the purified 3D protein was used to immunize mice following standard procedures for the production of monoclonal IgG antibodies. Two 3D protein-specific monoclonal IgG antibodies (hybridoma cell line 3D-2A10-IgG and hybridoma cell line 3D-3A 12-IgG) were prepared by conventional hybridoma technology. The corresponding hybridoma cell line has been deposited with the China Center for Type Culture Collection (CCTCC), CCTCC NO: c2014143 is hybridoma cell strain 3D-2A10-IgG and CCTCC NO: c2014141 is hybridoma cell strain 3D-3A12 IgG antibody. The preservation unit: china center for type culture Collection, the preservation Address Hubei Wuhan university, the preservation date: 7 month and 22 days 2014. The monoclonal IgG antibody may be an antibody comprising an Fc domain, a single chain antibody, or a Fab fragment. The definition and production of various antibodies is well known.

The hybridoma cell lines of 3D-2A10-IgG and 3D-3A12-IgG are subjected to subtype conversion to obtain two corresponding 3D protein specific monoclonal immunoglobulin A antibodies, a hybridoma cell strain 3D-2A10-IgA and a hybridoma cell strain 3D-3A 12-IgA. The corresponding hybridoma cell line has been deposited with the China Center for Type Culture Collection (CCTCC), CCTCC NO: c2014144 is hybridoma cell strain 3D-2A10-IgA and CCTCC NO: c2014142 is a hybridoma cell strain 3D-3A12-IgA antibody. The preservation unit: china center for type culture Collection, the preservation Address Hubei Wuhan university, the preservation date: 7 month and 22 days 2014.

Using recombinant 3D proteins deleted consecutively from either end, antibody-bound polypeptides were identified. Binding of 3D-3A12 IgA antibody to KEPAVLTS (SEQ ID NO: 3), 3D-2A10-IgA binding YSTYVKDELRSLDKI (SEQ ID NO: 9) is predicted from the binding specificity of the corresponding IgG antibody. Using sequence alignment, the identified polypeptides are highly conserved among all enterovirus strains from enterovirus subtypes a, B, C and D. As shown in Table 1 below, the 3D-3A12 IgA antibody binds to the consensus sequence KEPAVLX₇X₈The polypeptide (SEQ ID NO: 1) represented by (I) wherein X is₇Selected from T, H, R and N, X₈Selected from S, N and K. The 3D-2A10-IgA antibody binds to a consensus sequence X₁X₂TX₄VKDELRSX₁₂X₁₃KX₁₅Is represented by a polypeptide (SEQ ID NO: 2) wherein X₁Selected from Y, M, L and F, X₂Selected from S and V, X₄Selected from Y and F, X₁₂Selected from L, A, K and R, X₁₃Selected from D, E, T and S, X₁₅Selected from I and V.

TABLE 1 consensus sequences and exemplary sequences recognized by 3D-2A10-IgA or 3D-3A12 IgA antibodies

Enteroviruses include subtypes a, B, C and D.

The enterovirus a subtype contains 23 serotypes, exemplary 3D sequences include: (1) human enterovirus type 71 (EV 71), BrCr strain (GenBank AB 204852.1) (SEQ ID NO: 15), wherein SEQ ID NO: 15 comprises an antibody binding polypeptide consisting of SEQ ID NOS: 3 and 9 respectively, (2) human coxsackievirus A16 type strain shzh00-1 (GenBank AY790926.1) (SEQ ID NO: 16), wherein SEQ ID NO: 16 comprises an antibody binding polypeptide consisting of SEQ ID NOS: 3 and 9 respectively.

The enterovirus B subtype contains 60 serotypes, exemplary 3D sequences include: (1) human coxsackievirus B3Beijing0811 strain (GenBank GQ 141875.1) (SEQ ID NO: 17), wherein SEQ ID NO: 17 comprises an antibody binding polypeptide consisting of SEQ ID NOS: 5 and 10, respectively, (2) a human coxsackie A9 GRIGGS strain (GenBank D00627.1) (SEQ ID NO: 18), wherein SEQ ID NO: 18 comprises an antibody binding polypeptide consisting of SEQ ID NOS: 5 and 10 respectively.

An enterovirus subtype C contains 23 serotypes, exemplary 3D sequences include: (1) human poliovirus 1 strain CHN-Jiangxi// 89-1 (GenBank: AF 111984.2) (SEQ ID NO: 19), wherein SEQ ID NO: 19 comprises an antibody binding polypeptide consisting of SEQ ID NOS: 6 and 11 respectively, (2) a human coxsackievirus A1 KS-ZPH 01F/XJ/CHN/2011 isolate (GenBank: JX 174177.1) (SEQ ID NO: 20) wherein SEQ ID NO: 20 comprises an antibody binding polypeptide encoded by SEQ ID NOS: 6 and 12 respectively.

The enterovirus D subtype contains 5 serotypes, exemplary 3D sequences include: (1) human enterovirus 68 Fermon strain (GenBank: AY 426531.1) (SEQ ID NO: 21), wherein SEQ ID NO: 21 encompasses antibody-binding polypeptides encoded by seq id NOS: 7 and 13, respectively, (2) a human enterovirus 94E 210 isolate (GenBank: DQ 916376.1) (SEQ ID NO: 22), wherein SEQ ID NO: 22 comprises an antibody binding polypeptide consisting of SEQ ID NOS: 8 and 14 respectively.

The recombinant 3D protein can be used as an immunogen in immunogenic compositions, either alone or together with other proteins from enteroviruses. As shown in the examples below, the use of the EV 713D protein with bacterial flagellins induced an antibody response and partially protected challenge. Since the 3D protein is highly conserved in all enteroviruses, it is expected that the 3D protein can be used as an immunogen with broad protection against various subtypes. Furthermore, certain variants of the 3D protein may also be used in immunogenic compositions, wherein the variants have at least 85%, preferably 90%, more preferably 95% identity to the corresponding wild-type protein. Furthermore, the adjuvant may be any known, such as M59, aluminum adjuvant.

Since antibodies against 3D proteins do not show ADE effects, it is advantageous to use antibody compositions to treat enterovirus infections in patients. Antibody compositions for therapeutic use are well known, as are antibodies for the treatment of cancer. Antibody compositions typically comprise a pharmaceutically acceptable ingredient, such as sodium chloride dissolved in a pharmaceutically acceptable solution. The present invention demonstrates that 3D-2A10-IgA and 3D-3A12-IgA are able to partially protect the host from challenge with a survival rate of 60% (FIG. 11).

Examples

The following examples are provided for the sole purpose of illustrating the principles of the present invention; they are in no way intended to limit or otherwise narrow the scope of the present invention.

Example 1

EV 713D specific IgG monoclonal antibodies.

1.1 antigen preparation

The complete 3D gene of the strain EV71 BrCr (encoding the protein shown in SEQ ID NO: 15) was cloned into the vector pET28a at NcoI and XhoI. Briefly, E.coli BL21 (DE 3) containing recombinant 3D expression vector transformation was grown bacterially, these recombinant proteins were prepared by induction and purified by affinity chromatography on Ni-NTA columns (Qiagen).

1.2 immunization and preparation of EV 713D-specific IgG monoclonal antibody

The preparation of EV 713D-specific Monoclonal antibodies was as previously described (Li YM, Liu F, Han C and Yan HM. Monoclonal antibody blocks of the Toll-like receptor 5 binding formation of flagellin. Hybridoma (Larchmt) 2012 Feb;31(1): 60-62). Briefly, 5-week-old female SPF BALB/c mice were immunized subcutaneously with 100 μ g of 3D at 2-week intervals. Four weeks after the last boost and the first 3 days of cell fusion, mice were boosted with 200 μ g of 3D intraperitoneal inoculum. Three days later, mouse splenocytes were harvested and fused with SP2/0 using 50% polyethylene glycol (Sigma-Aldrich, Mo.). Hybridoma culture supernatants were screened by ELISA. Positive hybridoma cells were cloned by limiting dilution and stable hybridoma clones were injected into the peritoneal cavity of liquid paraffin-pretreated BALB/c mice. Subsequently, the monoclonal antibody was harvested and purified using an antibody purification kit (NAb)^TMProtein A/GSpin Kit, Thermo Scientific, USA), purified according to the manufacturer's instructions.

1.3 verification of binding specificity of 3D-specific IgG monoclonal antibodies

Vero-1008 cells in 24-well plates were infected with EV71 (MOI = 0.1). 24 hours after infection, the cells were fixed with absolute methanol and subjected to an indirect immunofluorescence assay (IFA) using 3D-specific monoclonal antibodies (3A 12, 2A10, 7A6G1 and 11F 1) followed by fluorescein isocyanate-conjugated goat anti-mouse IgG antibodies, flagellin-specific monoclonal antibody (5G 10) as a negative control and 3D-immunized mouse serum as a positive control. As shown in fig. 1 (a), 3a12, 2a10, 7A6G1 and 11F1 showed significant staining, reaching a level comparable to the positive control, and the negative control monoclonal antibody 5G10 showed no staining.

Vero-1008 cell culture and infection are described above. Cell lysates were separated by SDS-PAGE and transferred to PVDF membrane and blotted with indicated antibodies following conventional immunoblotting procedures. As shown in FIG. 1 (B), lanes 1, 3A12, 2A10, 3, 7A6G1, 4,11F1, 5G10 were used as a negative control, and lane 6, a positive control. The results showed that 3D protein specific IgG monoclonal antibodies (3 a12, 2a10, 7A6G1 and 11F 1) showed specific bands corresponding to the 3D protein, but the negative control (5G 10) showed no binding of 3D.

1.4 intracellular inhibition of EV71 replication by 3D-specific IgG monoclonal antibodies

Vero-1008 cells were seeded in 24-well plates at 1X 10 per well⁵Cells, after 24 hours in culture, were infected with EV71 at MOI = 0.1. After 1 hour of infection, cell supernatants were removed and cells were washed 3 times. Mu.g IgG was added to 100. mu.l OPTI-MEM, then 5. mu.l liposome 2000 was added to 100. mu.l OPTI-MEM, and finally the mixture was added to virus-infected cells. After 3 hours of transfection, cell supernatants were replaced with 0.5ml DMEM containing 3% FBS. Eight hours later, EV71 infected cells were harvested, and after 1 freeze-thaw cycle, viral titers (represented by PFU/well) were determined in these cell samples by plaque assay. As shown in fig. 2, transfected 3a12 and 2a10 significantly reduced viral titer, transfected 7A6G1 reduced viral titer but was less significant than 3a12 and 2a10, but transfected 11F1 failed to reduce viral titer. As for EV-5, this is a VP 2-specific IgG monoclonal antibody, transfected EV-5 failed to reduce viral titers, and it is noted that EV-5, when added directly to cell cultures, showed increased viral titers, indicating the effect of antibody-dependent enhancement (ADE). Data on viral titers indicate that the epitopes recognized by the different 3D-specific IgG monoclonal antibodies are important for their ability to inhibit EV71 replication.

Culture, infection and antibody treatment of Vero-1008 cells were as described above, except for the dose of antibody. As shown in fig. 2 (B), the reduction of viral titers by transfected 2a10 at the tested dose (0, 0.2%, 1 or 5 μ g/well) showed dose-dependent inhibition, but transfected EV-5 was not inhibited at all tested dose ranges. Three independent experiments were repeated and representative data were reported.

1.5 in vitro 3D polymerase activity inhibition by 3D specific IgG monoclonal antibodies

FIG. 3 (A) illustrates 3D (RdRp) -mediated RNA extension.

In a 10. mu.l reaction system (50 mM HEPES, 75 mM KCl, 5 mM MgCl)₂4 mM TCEP, 300. mu.M NTP, and 4. mu.M RNA complete), addition of 1 microgram of functional 3D triggers initiation and extension of RNA. RNA species were separated by electrophoresis on a 15% polyacrylamide gel containing 7 moles of urea and stained with Stains-All. To determine inhibition, 2 μ g of IgG monoclonal antibody was added to each reaction. The 3D RNA extension activity was determined by the appearance of an extended RNA band. As shown in FIG. 3 (B), lane 1, negative control without polymerase, lane 2, positive control group (no intervening factors), lane 3, 3A12, lane 4, 2A10, lane 5, 7A6G1, lane 6,11F1, and lane 7, EV-5. 3A12, 2A10 and 7A6G significantly inhibited the appearance of RNA extension bands, but 11F1 and EV-5 failed to inhibit RNA extension. Three separate experiments were performed and representative data are reported.

1.6 antiviral Effect of 3D specific monoclonal antibodies in murine models

The antiviral efficacy of EV 713D-specific IgG was studied in a murine model. 30 1-day-old newborn mice were randomly divided into 6 groups (5 mice per group). 100. mu.g/50. mu.l of 3A12, 2A10, 7A6G1, 11F1 or EV-5 IgG antibody was administered to each group by intraperitoneal inoculation, and PBS group was used as a negative control, and 10 mice were administered to each group by intraperitoneal inoculation³TCID₅₀EV71 challenge. IgG was injected 4 times at 24 hour intervals. Mice survival data were collected daily for 2 weeks. As shown in FIG. 4, 2A10-IgG and 3A12-IgG provided 20% or 40% protection, respectively.

1.7, 2A10-IgG or EV-5-IgG in vitro

Will be 1 × 10⁴PFU EV71 was added to 200. mu.l of monoclonal antibody (EV-5 antibody against EV71VP2, or 2A10 IgG antibody against EV 713D) in serial dilutions. After 1 hour incubation, the mixture infected Caco-2 cells. EV71 infected Caco-2 cells were collected and virus titers were determined in samples of these cells by plaque assay. As shown in FIG. 5, at a concentration of 0.25-16. mu.g/ml, from baseline (P)<0.01) compared to EV-5, but 2a10 did not enhance viral infection at all doses tested.

1.8 mapping of EV 713D-specific epitopes of 3A12 and 2A10 and the spatial conditions in a three-dimensional model of EV 713D (1 RA 6)

The 3D gene (encoding the protein represented by SEQ ID NO: 15) and its truncated mutants were cloned into pET28a expression vector, and the binding activity of IgAs to the 3D proteins and mutants was determined by Western blotting. In addition, the exact range of recognition of 3A12-IgG and 2A10 IgG antibodies was determined using synthetic polypeptides of varying lengths. As shown in table 1, the polypeptides identified by 3a12 and 2a10 are: KEPAVLTS (SEQ ID NO: 3) and YSTYVKDELRSLDKI (SEQ ID NO: 9). Alignment of various strains of enteroviruses from all subtypes A, B, C and D revealed a consensus sequence KEPAVLX recognized by 3A12₇X₈(SEQ ID NO: 1), and another consensus sequence X recognized by 2A10₁X₂TX₄VKDELRSX₁₂X₁₃KX₁₅(SEQ ID NO: 2). As shown in fig. 6, EV 713D (1 RA 6) was used to show the positions of the two recognized epitopes of 3a12 and 2a 10.

Example 2

EV 713D protein specific monoclonal immunoglobulin A antibody

2.1, 3D protein-specific monoclonal immunoglobulin A antibodies

The 2A10-IgA and 3A12-IgA monoclonal antibodies are derived from the 2A10-IgG and 3A12-IgG monoclonal antibodies, respectively, and the subtype switch is by conventional subtype switching techniques.

2.2 intracellular staining of 3D proteins by monoclonal immunoglobulin A antibodies specific for the 3D proteins

VERO-1008 cells were plated in 24-well plates and infected with EV71 (MOI = 0.1). 24 hours after infection, cells were fixed with methanol and subjected to indirect immunofluorescence staining (IFA), using 3D protein-specific monoclonal immunoglobulin A antibodies 3A12-IgA and 2A10-IgA, 16CF7-IgA (anti-MeV) as negative controls, and sera of 3D-immunized mice as positive controls. As shown in FIG. 7 (A), 3A12-IgA and 2A10-IgA, as well as positive sera, were able to react with EV71 infected cells, but the negative control showed no staining.

2.3 immunoblotting of monoclonal immunoglobulin A antibodies specific for 3D proteins

EV 71-infected VERO-C1008 cells were lysed and the cell lysates were separated on SDS-PAGE and transferred to PVDF membrane, immunoblotted with 3A12-IgA, 2A10-IgA, 16CF7-IgA, and positive sera. As shown in FIG. 7 (B), 3A12-IgA, 2A10-IgA and positive sera stained specifically EV 71-infected cells, but 16CF7-IgA did not. These results demonstrate that 3A12-IgA and 2A10-IgA retained the 3D specificity of the corresponding 3A12-IgG and 2A 10-IgG.

2.4 EV 713D-specific IgA inhibits EV71 replication intracellularly by pIgR

(i) Polarized VERO-pIgR cells, containing 0.4 m membrane in the Transwell culture; infection with EV71 allowed the virus to attach to the cells (cell adhesion). After 1 hour, the top of the cells and both sides of the substrate were washed to remove the virus that was not adsorbed. Then, 400 μ l of complete DMEM was added to the apical side edge; 3D-specific or non-relevant IgA antibodies (30. mu.g/100. mu.l) were added to the base side. After 24 hours, both sides of the cells were washed well with PBS, 400. mu.l of complete DMEM was collected, and the virus titer in the cell samples was determined by plaque assay. As shown in FIG. 8 (A), 3A12-IgA and 2A10-IgA were able to significantly inhibit EV71 replication, but 16CF7-IgA was not inhibited.

(ii) Polarized VERO-pIgR cells were manipulated as (i) but with different amounts of IgA antibodies (3A 12-IgA and 16CF 7-IgA). As shown in fig. 8 (B), 3a12-IgA inhibited EV71 replication intracellularly, exhibiting dose-dependence.

(iii) Polarized VERO-pIgR cells were manipulated as (i) but with the corresponding IgG antibodies (3A 12-IgG, 2A10-IgG and 16CF 7-IgG). As shown in fig. 8 (C), all IgG antibodies had no significant inhibitory effect on EV71 replication.

(iv) The procedure was as in (i), but VERO-pIgR cells were exchanged for VERO-1008 cells. As shown in fig. 8 (D), all three IgA antibodies (3 a12-IgA, 2a10-IgA and 16CF 7-IgA) had no significant inhibitory effect on EV71 replication, indicating that IgA-mediated intracellular inhibition of EV71 replication is dependent on pIgR expressed on the VERO cell surface.

2.5 EV 713D specific IgAs inhibit the replication of EV71 and CV strains intracellularly

Experiments to inhibit replication of EV71 and CV strains intracellularly are described in example 2.4. The results are summarized in table 2 below.

TABLE 2 intracellular inhibition of replication of EV71 and CV strains

Where "a" indicates that "% virus reduction" was calculated after calculating the percentage of virus survival in the presence of different antibodies using the virus titer of the medium group as 100%.

2.6 EV 713D IgA inhibits viral protein expression

(i) Polarized VERO-pIgR cells, containing 0.4 m membrane in the Transwell culture; infection with EV71 allowed the virus to attach to the cells (cell adhesion). After 1 hour, the top of the cells and both sides of the substrate were washed to remove the virus that was not adsorbed. Then, 400 μ l of complete DMEM was added to the apical side edge; 3D-specific or non-relevant IgA antibodies (30. mu.g/100. mu.l) were added to the base side. After 24 hours, both sides of the cells were washed well with PBS, 400. mu.l of complete DMEM was collected, and the amounts of 3D and VP2 proteins in these cell samples were determined by immunoblotting (WB). As shown in FIG. 9 (A), 3A12-IgA (lane 1) and 2A10-IgA (lane 2) significantly inhibited the expression of 3D and VP2, but 16CF7-IgA (lane 3) and DMEM (lane 4) did not significantly inhibit.

(ii) Polarized VERO-pIgR cells were manipulated as in (i). Cell samples were analyzed by ELISA. As shown in FIG. 9 (B), 3A12-IgA and 2A10-IgA significantly inhibited the expression of 3D.

2.7, 3A12-IgA and 2A10-IgA inhibit the RNA extension activity of 3D polymerase

In a 10. mu.l reaction system (50 mM HEPES, 75 mM KCl, 5 mM MgCl)₂4 mM TCEP, 300. mu.M NTP, and 4. mu.M RNA complete), 1 microgram of functional 3D was added, triggering initiation and extension of the RNA. RNA species were separated by electrophoresis on a 15% polyacrylamide gel containing 7 moles of urea and stained with Stains-All. As shown in FIG. 10, lane 1, negative control without polymerase, lane 2, positive control group (no intervening factors), lane 3, 16CF7-IgA (2. mu.g); lane 4, 2A10-IgA (2. mu.g), lane 5, 3A12-IgA (2. mu.g). Three separate experiments were performed and representative data are reported.

2.8 evaluation of the antiviral efficacy of EV 713D-specific IgA antibodies in the neonatal mouse model

1 day old newborn mice were administered 100. mu.g/50. mu.l IgA antibody by intraperitoneal inoculation, and then 10 days old newborn mice were administered per group by intraperitoneal inoculation³TCID₅₀The EV71 mouse was adapted to strain challenge. IgA antibodies were injected 4 times at 24 hour intervals. Mice survival data were collected daily for 3 weeks. As shown in FIG. 11, 2A10-IgA and 3A12-IgA provided 40% or 60% protection, respectively.

Example 3

3D as antigen

3.1 identification of 3D in recombinant vaccinia Virus Tiantan Strain (VTT-3D) expressing 3D

EV 713D was detected in VERO-1008 cells infected with recombinant vaccinia virus. As shown in FIG. 7, lane 1, protein molecular weight marker, lane 2, mock-infected VERO-1008 control, lane 3, VTTenv-infected VERO-1008, lanes 4-7, 4 purified recombinant vaccinia virus clone-infected VERO-1008. Infected or uninfected VERO-1008 was separated by SDS-PAGE and transferred to PVDF membrane. The 3D-specific IgG monoclonal antibody 2a10 IgG antibody was used as the binding antibody. The results show that the 3D gene was inserted into the vaccinia virus genome in VERO-1008 cells and was correctly expressed.

3.2 immunization procedure

TABLE 3 immunization procedure

Figure 13 is a schematic of an immunization protocol.

Antibody responses were detected after each immunization: first reinforcement (fig. 14), first reinforcement (fig. 15), and second reinforcement (fig. 16). Each mouse in the VTT-3D group was immunized with 10 microliters of 100 microliters⁷PFU virus, whereas each mouse in the 3D group was immunized with 100 μ l of 30 μ g 3D protein. Serum and mucosal samples from vagina and saliva were collected and titers of IgA against 3D protein determined by ELISA.

Antibody responses of the newborn mice tested (fig. 17).

3.3 EV71 poison challenge protection

After three immunizations, each neonatal mouse was challenged with 10 in VTT-3D, PBS and EV71 inactivated groups³TCID₅₀A virus. Newborn mice were observed daily. Immunization with inactivated EV71 fully protected the mice. All mice in the PBS negative control group died 3-5 days after challenge. As shown in fig. 18, 3D and VTT-3D provide 10% -30% protection.

Example 4

4.1 immunization with purified 3D proteins

Expression and purification of the 3D protein is described in example 1.1. The mice were then immunized with the pure 3D protein plus flagellin or CTB from e.coli as adjuvant by Subcutaneous (SC), Intranasal (IN) or Intraperitoneal (IP) routes. Table 4 summarizes the immunization protocol. The interval between two immunizations was 2 weeks, the volume of SC and IP was 100. mu.l, and the volume of IN was 20. mu.L.

TABLE 4 immunization protocol with purified 3D proteins

Group of	Immunity composition	Immunization method	Number of mice per group
				1	30μg3D+5μgKF	2xSC+2xIN	6
2	30μg3D+5μgKF	2xIP+2xIN	6
				3	30μg3D+2μgCTB	2xSC+2xIN	6
4	30μg3D+2μgCTB	2xIP+2xIN	6
				5	PBS	2xSC+2xIN	6

3.2, antibody response

After 2 weeks of the final immunization, serum, small intestine and lung samples, and homogenates of small intestine and lung samples were collected and supernatants were collected for testing. The 3D specific IgA or IgG antibody responses in these samples were titrated by ELISA. Figure 19 shows 3D-specific IgG antibody titers: serum (a), lung (b) and small intestine (c); titer of 3D-specific IgA antibody: serum (d), lung (e) and small intestine (F).

While the invention will be described with reference to specific embodiments, it will be understood that the examples are illustrative and that the scope of the invention is not limited thereto. Alternative embodiments of the invention will become apparent to those of ordinary skill in the art to which the invention relates. Such alternative embodiments are to be considered as included within the spirit and scope of the present invention. The scope of the invention is, therefore, indicated by the appended claims, and is supported by the foregoing description.

Claims

1. A monoclonal immunoglobulin IgA antibody, wherein the monoclonal IgA antibody specifically binds to a polypeptide, wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 or SEQ ID NO: 2, a consensus sequence representation; and SEQ ID NO: 1 is a monoclonal IgA antibody combined by the polypeptide with the preservation number of CCTCC NO: c2014144, and the hybridoma cell line 3D-2A10-IgA produced by the method and has the same sequence as SEQ ID NO: 2 is a monoclonal IgA antibody combined by the polypeptide with the preservation number of CCTCCNO: c2014142 hybridoma cell line 3D-3A12-IgA produced.

2. The monoclonal IgA antibody of claim 1, wherein SEQ ID NO: 1 is a polypeptide selected from the group consisting of SEQ ID NOS: 3-8, SEQ ID NO: 2 is selected from the group consisting of SEQ ID NOS: 10-14.

3. A composition comprising at least one first monoclonal IgA antibody that specifically binds to a polypeptide consisting of the consensus sequence of SEQ ID NO: 1, at least one second monoclonal IgA antibody, specifically binds to a polypeptide represented by the consensus sequence SEQ ID NO: 2, and a pharmaceutically acceptable solution, wherein the second polypeptide has a sequence similar to SEQ id no: 1 is a monoclonal IgA antibody consisting of a sequence with a collection number of CCTCC NO: c2014144, and the hybridoma cell line 3D-2A10-IgA produced by the method and has the same sequence as SEQ ID NO: 2 is a monoclonal IgA antibody having a deposit number of CCTCC NO: c2014142 hybridoma cell line 3D-3A12-IgA produced.

4. The composition of claim 3, wherein the first polypeptide is a polypeptide selected from the group consisting of SEQ ID NOS: 3-8, and the second polypeptide is a polypeptide selected from the group consisting of SEQ ID NOS: 10-14.